Myo-inositol has been widely utilized as a component of nutritional foods, feeds, pharmaceuticals, and the like since it is an essential substance for many higher animals. For example, myo-inositol is known to play an important role in metabolism of fats and cholesterols and is held to be effective in prevention and treatment of hypercholesterolemia and the like. Many improvements of industrial-scale production processes for myo-inositol have therefore been proposed.
In the past, myo-inositol was extracted directly from rice bran, corn steep liquor, and the like. In addition to giving a low yield of myo-inositol, the extraction method produces many impurities, making purification of myo-inositol difficult and leading to very low production efficiency. Consequently, a method for producing myo-inositol from the culture obtained by culturing Saccharomyces cerevisiae having the ability to produce myo-inositol was also proposed. Nonetheless, this method was not implemented on an industrial scale as the productivity was still low and the method was not economically viable.
Microorganisms that can produce inositol more efficiently have therefore been sought. Patent Reference 1 discloses the discovery of yeast of the genus Candida capable of secreting inositol extracellularly and the utilization thereof. Patent References 2 and 3 disclose introduction of mutations to impart resistance to glucose antimetabolites and antibiotics, respectively, into the above yeast of the genus Candida. Patent References 4, 5, and 6 disclose improving the yield of inositol by introducing mutations to impart resistance to tertiary amines, hexachlorocyclohexane, and cetyl trimethylammonium salt, respectively, into yeasts of the genus Candida having the ability to produce inositol. Patent Reference 7 similarly discloses introduction of a mutation to impart resistance to 6-halogeno-6-deoxyglucose into yeast of the genus Candida having the ability to produce inositol. Patent Reference 8 also discloses introduction of a mutation to impart resistance to halogenated pyruvic acid into yeast of the genus Candida having the ability to produce inositol.
The prior art also describes transformation of yeast by gene recombination. Patent Reference 9 discloses that it is possible to impart the ability to produce inositol to yeast by transforming yeast of the genus Candida that does not have the ability to secrete inositol by inositol-1-phosphate synthase-encoding DNA alone, based on the reasonable inference that inositol-1-phosphate synthase is responsible for a rate-limiting reaction in the series of myo-inositol biosynthetic reactions. Patent Reference 10 discloses that the inositol productivity of yeast is improved by introducing inositol-1-phosphate synthase-encoding DNA alone into yeast under the control of a glycerol-3-phosphate dehydrogenase gene promoter.
Patent Reference 11 relates to inositol production by methanol-assimilating yeast Pichia pastoris, and it discloses introduction of an inositol phosphate phosphatase gene simultaneously with introduction of a myo-inositol-1-phosphate synthase gene into this yeast. However, the significance and effect of the additional introduction of an inositol phosphate phosphatase gene are not revealed.
Therefore, all of the above references relating to yeasts presuppose that inositol-1-phosphate synthase is responsible for a rate-limiting reaction in the series of myo-inositol biosynthetic reactions and do not suggest the importance of other enzymes that are present in the myo-inositol biosynthetic pathway.
On the other hand, prokaryotic microorganisms typified by Escherichia coli are extremely attractive organisms for industrial production as compared to yeasts due to strong proliferative capacity and various superiorities in fermentation control. However, no prokaryotic microorganism having an endogenous myo-inositol biosynthetic pathway is known.
Therefore, for the production of myo-inositol in an industrial scale by prokaryotic microorganisms, it is essential to construct an exogenous biosynthetic pathway within a prokaryotic microbial host.
Specifically, the following catalytic activities are necessary to construct a functional myo-inositol biosynthetic pathway within a prokaryotic microbial host:
activity 1: an activity to produce glucose-6-phosphate from a suitable carbon source;
activity 2: an activity to convert glucose-6-phosphate into myo-inositol-1-phosphate, that is, inositol-1-phosphate synthase activity; and
activity 3: a phosphatase activity utilizing myo-inositol-1-phosphate as a substrate.
However, since glucose-6-phosphate that is a product of activity 1 is a metabolic intermediate universally produced by prokaryotic microorganisms, it is not essential to impart this activity to prokaryotic microorganisms. With regard to activity 3 as well, as far as the inventors know, the majority of prokaryotic microbial host cells that are suited to industrial production by conventional gene recombination techniques express endogenous inositol monophosphatase, or they have general monophosphatase activity capable of using myo-inositol-1-phosphate as a substrate. On the other hand, as for activity 2, there are many examples of prokaryotic microorganisms that do not have an inositol-1-phosphate synthase gene. Inositol-1-phosphate synthase is believed to be responsible for a rate-limiting reaction in myo-inositol biosynthetic reactions, as was mentioned above. Therefore, the introduction of an inositol-1-phosphate synthase gene into the cell has been thought to be necessary and sufficient to construct a functional myo-inositol biosynthetic pathway within a prokaryotic microbial host.
In fact, Non-patent Reference 1 discloses myo-inositol production within E. coli transformants, but only an inositol-1-phosphate synthase gene is introduced into these transformants.
Patent Reference 12 also discloses that only an inositol-1-phosphate synthase gene is introduced into an E. coli host cell to produce transformants thereby constructing an exogenous myo-inositol biosynthetic pathway within the transformants. However, the final target product in this reference is D-glucaric acid; myo-inositol is produced as an intermediate. It is noteworthy that this reference also states: “It should also be noted that we did not overexpress the suhB gene or a homologous phosphatase. However, no myo-inositol-1-phosphate was detected among the culture products, while myo-inositol did accumulate. Therefore, we conclude that the phosphatase activity is not limiting flux through the pathway” (page 33, lines 2-5).
Therefore, the prior art neither discloses nor suggests a critical role for inositol monophosphatase in myo-inositol production by recombinant microorganisms.